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  1. Thank you for viewing this presentation. • We would like to remind you that this material is the property of the author.It is provided to you by the ERS for your personal use only, as submitted by the author. • 2012 by the author

  2. From traditional bacteriology to rapid molecular methods: the revolution is going on (September 2012; Vienna) Keertan Dheda, MB.BCh, FCP(SA), FCCP, PhD (Lond), FRCP (Lond) Professor and Head: Lung Infection and Immunity Unit Division of Pulmonology & UCT Lung Institute, Department of Medicine, email: keertan.dheda@uct.ac.za Conflict of interest: none

  3. “If TB and AIDS are a snake, then the head is in South Africa while the tail is quickly moving through other African countries… And if the head of the snake is in South Africa then the teeth are in Durban” Dr Aaron Motsaledi (SA minister of Health) World TB Day, 24 March 2011 1 in 100 South Africans have TB 500 000 cases per annum (1000 new cases every day!)

  4. Overview • Epidemiology of DR-TB, TB control, and the unmet need for diagnostics • Appreciating phenotype and sampling error and its implications • NAATS: Gene Xpert MTB-RIF Hain MDR TB plus (including new version) Hain MDR TB sl Plex ID (combination of mass spec and NAAT) • Newer and novel approaches: HRM assay MDR on a chip (array and microfuidics) Sequencing Lateral flow assays • Summary and conclusions

  5. What is the size of the problem globally? • Worldwide 650 000 prevalent cases of MDR-TB in 2010 (5·4% of the 12 million prevalent cases) (490 000 new cases; 3.4% of new cases and 20% of retreatment cases) WHO. Global TB Control. 2011. • XDR-TB: globally~ 25000 XDR-TB cases annually

  6. MDR-TB in SA • 80% of MDR-TB results from ongoing transmission Streicher and Warren, Infect Gen Evol, 2011 • In Khayelitsha, Cape Town, 81% of MDR-TB likely due to primary transmission Cox HS, PLoS One, 2010 • Therefore any cost saving strategy that targets testing, only to those with risk factors, will miss a substantial number of cases and fail to supress on-going transmission

  7. N= 46 isolates from the WC Genotyping and mutational analysis = 81% acquired resistance

  8. N= 199 XDR-TB

  9. Major unmet diagnostic need • Only 7% MDR-TB reported and 1-2% actually treated to WHO standards • Less than 2% of cases have available DST result • Very few reference laboratories in the 22 high burden countries • Need user-friendly rapid tools, preferably at point of care, given decentralised MDR treatment programmes

  10. Approach to diagnosis Visualise the bug- LED microscopy, various techniques to concentrate the bugs (AB, magnets, spin filters) Pai et al. Sem Resp Crit Care Med 2008

  11. Approach to diagnosis and new technologies Grow the bug- liquid culture, MODS Pai et al. Sem Resp Crit Care Med 2008

  12. Diagnostic test impact Diagnostic only one element of DR-TB control

  13. TB diagnosis and control in HIV-infected persons • ~ 20% are sputum scarce • 20% have extrapulmonary TB (paucibaciliary) • ~ 30 to 40% are smear negative- often poor quality • Smaller volume of sputum and lower concentration of M.tb • Pulmonary infiltrates atypical or chest x-ray may be normal LOW CD4 count < 200 cells/ ml Have an extended length of stay before diagnosis is made and treatment is initiated Many patients die undiagnosed with TB Theron and Dheda, AJRCCM, 2011

  14. Problem of sample aquisition • Even with tests like Xpert about 1/3 of TB will probably remain undiagnosed without additional interventions (sputum scarce PTB, undiagnosed fraction of smear negative TB, and EPTB) Sputum induction and bronchoscopy- only available in tertiary care facilities

  15. What is Xpert MTB/RIF? • Xpert is an automated real-time PCR for the diagnosis of TB and genotypic rifampicin resistance • WHO approved: frontline dx for individuals suspected of TB-HIV co-infection • SA DoH has initiated the replacement of smear by Xpert for TB suspects Scott, PLoS Med, 2011

  16. Boehme C, NEJM, 2010

  17. C Boehme, FIND Diagnostics

  18. Gene Xpert (WHO endorsed) • Cost: R1003 (Path Care)- Jan 2012 R 80 per cartridge (excludes capital cost and labour) • How good is it: Sensitivity= 97% in smear +ve and 70% in smear negative; Specificity = 99% User-friendly and quick- POC. Closed system. However, only R resistance (not I, FQ, Cap, Etham like Hain) Currently being rolled out in SA

  19. How does Xpert MTB/RIF perform? Boehme et al, NEJM, 2010 (N= 1730); Boehme et al, Lancet, 2011 (n= 5000) TB Rif R

  20. Gene Xpert (WHO endorsed) • Drawbacks: (i) Expensive. (iii) Negative test high rule-out value in uninfected but not HIV-infected persons. Theron and Dheda, AJRCCM, 2011 (iii) PPV for DR-TB locally is sub-optimal so may be overcalling DR-TB- new cartridge being trialled (G5).

  21. HIV-infected persons: Studies from ARV clinic in CT (n= 468 patients) • If no sputum- 2 induced samples • Otherwise 1 spot and 1 induced sample • 10% of cases no samples could be obtained Lawn SD, PLoS Med, 2011

  22. Studies from primary care TB clinics- Cape Town (n=480 with suspected TB) Theron and Dheda, AJRCCM, 2011

  23. Boehme C, Lancet, 2011Multisite evaluation studyComment by Theron, Lancet, 2011 (letter) • NPV was 91·3%(389/426; 95% CI 88·3–93·6) in HIV-infected versus 96·0%(748/779; 94·4–97·2;p<0·001) in the uninfected; the respective negative LRs were 0·18 (0·17–0·19) versus 0·09 (0·09–0·10). • Thus, about one in ten people that have active tuberculosis will have a negative test result.

  24. Studies from primary care TB clinic- Johannesburg (n= 311 TB suspects) Scott and Stevens, PLoS Med, 2011 Sensitivity in HIV-infected persons not significantly different from the CT study

  25. Xpert MTB/RIF research gapsBeyond diagnostic accuracy to patient outcomes

  26. Xpert RCT: Patient flow Hypothesis: One sputum Xpert MTB/RIF performed at the point-of-treatment is feasible and will improve TB-related morbidity and patient-level costs in individuals suspected of TB who present to primary level clinics in Africa

  27. TB-NEAT study- N= 508 participants in Durban and CT (of 600 participants) Xpert MTB/RIF sensitivity is significantly diminished in HIV infected vs. uninfected patients (p=0.04327)

  28. What currently available approaches can be used if Xpert is negative? • Perform CXR (rule out test) • Await culture • Perform a second Xpert • Refer for further investigation • Hain not an option in smear negative persons • Treat empirically for TB (commonest approach)

  29. How should Xpert be integrated with existing diagnostic algorithms? • Assessed the diagnostic accuracy and/or cost-effectiveness of smear-microscopy, chest-radiography, IGRAs combined with a single Xpert-MTB/RIF assay in 480 patients with suspected TB • Xpert negative- although CXR has poor rule-in value, it can reliably rule-out TB in approximately 1 in 4 of such cases. Theron and Dheda, Eur Resp J, 2011

  30. What currently available approaches can be used if Xpert is negative? • Perform CXR (rule out test) • Await culture • Perform a second Xpert (10% increase yield in SM-ve) • Refer for further investigation • Hain not an option in smear negative persons • Blood culture • Treat empirically for TB (commonest approach)

  31. Other applications of Xpert: Bacterial burden and infectiousness Theron, Peter and Dheda, AJRCCM, 2011

  32. Bacterial burden and infectiousness • Evaluated CT values in 496 patients with suspected TB • Xpert CT values have poor rule-in [cut-point ≤20.2; sensitivity 32.3%; specificity 97.1%] • Moderately good rule-out value for smear positivity [NPV 80.0%]. Thus, 20% of individuals with CT values >31.8 were smear- positive patients erroneously ruled out as smear-negatives. • But smear status a crude proxy of infectiousness and Xpert may also detect intact but dead bugs! • Same considerations apply to disease monitoring in MDR patients Theron, Peter and Dheda, Clin Infect Dis, 2011

  33. Urine-orientated approach in HIV-infected sputum-scarce persons? • Sputum-based diagnostic unhelpful in 20-30% HIV-infected patients • 116/242 culture +ve patients (54/116 i.e. 48% SS or SN) • 18% (20/116) of all culture +ve were sputum scarce • In this group (using urine) sensitivity: Xpert 40% (95%CI: 22-61) LAM 58% (95%CI: 49-67) LAM and Xpert 68% (95%CI: 60-77) [better than either; p= 0.003] In 6/14 +ve urine cases, LAM was exclusively positive Peter and Dheda, PLoS One, 2012

  34. Urine diagnostics to target HIV-infected sputum-scarce persons • Urine centrifugation and pelleting improved performance of Xpert in paired samples compared to unprocessed urine (42 vs 8%; p= 0.003) Urine-orientated approach feasible when a sputum-based tests are not feasible Peter and Dheda, PLoS One, 2012

  35. DST: Line probe assay (Hain) A • Hain Lifescience GenoType® MTBDRplus • Clinical samples (sens, spec): Rif (99; 99%) INH (85; 99)% Smear positive samples Morgan M, BMC Infect Dis, 2005; Ling D, Eur Resp J, 2008 Barnard M, AJRCCM, 2008

  36. A DST: Line probe assay (Hain, InnoLip A) • HainMTBDRplus (version 2.0) • In 104 smear-ve culture +ve sputum samples combined sensitivity (80%) and specificity (99%) • Crudu V, J Clin Micro, 2012 • Hain Lifescience GenoType® MTBDRplus • Clinical samples (sens, spec): Rif (99; 99%) INH (85; 99)% Smear positive samples Morgan M, BMC Infect Dis, 2005; Ling D, Eur Resp J, 2008 Barnard M, AJRCCM, 2008

  37. DST: Line probe assay (Hain) A • HainMTBDRplus (version 2.0) • In 104 smear-ve culture +ve sputum samples combined sensitivity (80%) and specificity (99%) • R100 per test in public sector and R571 in private sector • Crudu V, J Clin Micro, 2012

  38. Hain MDR+ sl version- to be used when R resistance is noted. Rapid evaluation of drug-resistance for FQ, AG + capreomycin [SLID] and ethambutol

  39. Hain MDR+ sl version- to be used when R resistance is noted. Rapid evaluation of drug-resistance for FQ, AG + capreomycin [SLID] and ethambutol Total 64 sputum samples ( 26 DR-TB)) FQ (89%; 8/9), AG/capreo (87%; 7/8), and ethambutol (39%; 10/26); 100% specificity Hilleman D, J Clin Micro, 2009 Miotto P, ERJ, 2012 (10 clinical samples resistant to SLIDS) Lancoma A, JCM, 2012 (52 clinical samples resistant to SLIDS)

  40. Hain MDR+ sl version Total 64 sputum samples (26 DR-TB) FQ (89%; 8/9), AG/capreo (87%; 7/8), and ethambutol (39%; 10/26); 100% specificity Hilleman D, J Clin Micro, 2009 Miotto P, ERJ, 2012 (10 clinical samples resistant to SLIDS) Lancoma A, JCM, 2012 (52 clinical samples resistant to SLIDS)

  41. 206 MDR or XDR culture isolates • Excellent sensitivity and spec in isolates • Markedly cuts down on time to diagnosis if patient is culture +ve

  42. OVERALL SENSITIVITY AND SPEC IN SMEAR +VE AND -VES • N= 158 sputum samples from patients with proven M and XDR-TB • All patients were culture positive

  43. Smear positive • Thus, MTBDRplus (version 2.0) very useful in smear +ve samples- make diagnosis of MDR immediately • TOTAL INDETERMINATES= 3.19%= • [9/19 (47.4%) indeterminate in scanty pos vs. 1/71 (0.01%) in 1+ to 3+ pos] • If MTBDRplus or Xpertpos proceed to the Hain SL in smear positive cases • TOTAL INDETERMINATES= 13%= • [9/19 (47%) in scanty pos vs. 11/74 (15%) in 1+ to 3+ pos] • Culture DST still required to clarify type of SLID resistance

  44. Smear negative • MTBDR PLUS STILL EXCELLENT Sens and Spec IN SMEAR NEG SAMPLES but 39% of results indeterminate! • In smear-ve samples 37% of SL were indeterminate • Thus, only useful in smear +ve samples

  45. Combined approaches: PCR and mass spec (alternative to Hain and Xpert for isolate ID and DST) • m • PCR for gene-specific mutations followed by R, I, E and FQ resistance; already commercially available

  46. Why do we need new tests? • Xpert is accurate & cost-effective but very costly (even at $10 per cartridge consume about 25% of SA NTP budget) - stable power supply - suited to centralised rather than decentralised use - in up to a third of cases diagnosis cannot be made (sputum scarce, EPTB, smear negative TB undiagnosed) • Large burden of undiagnosed TB (cost and access) • Lack of a cheap same day test (human aspect and poverty)

  47. POC detection technologies • NAAT with visual real-time readout including HRM • NAAT with lateral flow readout • NAAT with calorimetric readout (including LAMP) • NAAT with biosensor readout (electro-chemical • detection, piezoelectric quartz crystal biosensors, • magnetoeleastic biosensors) • NAAT with SERS readout • Biochip readouts for DR TB (currently not automated • and uses DNA from isolates) • Park H, JCM, 2006

  48. Several new commercial platforms that lend themselves to POC NAAT detection are now available http://www.iquum.com/products/analyzer.shtml

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